Effects of polycyclic aromatic hydrocarbons on the proliferation and differentiation of placental cells

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Methods HTR-8/SVneo cells were treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, which is used as a substitute for BPA-free products. After confirming the dose response for each reagent using the prepared cells, the cells were incubated for 24, 48, and 72 h. Cell viability was confirmed using the XTT assay. Each experiment was performed with the minimum number of samples (n = 3) required for statistical analysis. The results were analyzed using t-tests; p < 0.05 was considered statistically significant. Results After treatment with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, the absorbance measured using the XTT assay decreased significantly with increasing concentration. The absorbance decreased significantly over time following treatment with each endocrine disruptor at the concentration confirmed by the dose–response analysis. Conclusions This study showed that anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4-(9-fluorenylidene)diphenol—a BPA substitute—affect cell viability and necrosis in the placental cell line. The study indicates the serious effects of PAHs that negatively affect pregnancy but were previously unknown. Further, this study would serve as a reference for the identification of harmful PAHs during pregnancy prognosis in women who are more susceptible to PAH exposure. Endocrinology & Metabolism Pregnancy Placenta Endocrine disruptors Anthracene Female reproduction Polycyclic aromatic hydrocarbons Figures Figure 1 Figure 2 Background Endocrine disruptors are chemicals that can disrupt the endocrine system in both humans and animals. Female reproductive ability is an important process regulated by hormones and is susceptible to exposure to endocrine-disrupting substances. Endocrine disruptors have a benzene ring structure similar to that of steroid hormones among several female hormones. Therefore, they can easily affect reproductive hormones with a benzene ring structure [ 1 , 2 ]. Female reproductive disorders caused by endocrine disruptors can lead to inadequate hormone production, infertility, anovulation, premature ovarian failure, and menstrual disorders. Various endocrine disruptors have been reported to disrupt the endocrine system in reproductive-aged women and to cause congenital disabilities [ 3 , 4 ]. Polycyclic aromatic hydrocarbons (PAHs), which are endocrine disruptors, are common environmental pollutants released by incomplete combustion processes. PAHs emitted into our living environment are of interest because they have harmful side effects to humans, such as teratogenicity, mutagenicity, and carcinogenicity [ 5 ]. PAHs enter the body primarily through inhalation and ingestion. In pregnant women, PAHs that enter the body induce side effects on the mother, which are also transmitted through the placenta to the fetus, causing severe fetal developmental problems [ 6 ]. The growth and function of the placenta are regulated and coordinated to perform the placental role efficiently during pregnancy. If PAHs are transmitted from the mother to the fetus through the placenta, they negatively affect fetal growth. In addition, exposure to harmful substances such as PAHs during pregnancy can lead to disability even after the fetus grows into an adult [ 7 ]. A recent study analyzed the correlation between preterm labor and the concentration of PAHs in the placenta of women living in areas with high PAH exposure. This study showed that high PAH concentration, DNA products exposed to PAHs, and regulation of endocrine metabolic pathways affect preterm labor in pregnant women living near areas contaminated with environmental pollutants. Given the results of these studies, exposure to PAHs during pregnancy is a critical issue in terms of teratogenicity, fetal growth, and preterm labor. However, there are various PAH types, and the studies so far have mainly focused on bisphenol A (BPA) and some phthalates. Therefore, we experimented on the PAHs that have not been studied for their effects on pregnancy. We examined cell viability and necrosis in a placental cell line treated with the selected PAHs. This study indicates the serious effects of PAHs that negatively affect pregnancy but were previously unknown. Moreover, the study is a reference for the identification of harmful PAHs during pregnancy prognosis in women who are more susceptible to PAH exposure. Methods Chemicals and reagents Four PAHs, which have not been explored for their effects on pregnancy, were selected for experimentation on cell viability and necrosis in a placental cell line treated with the selected PAHs. In addition, 4,4-9(fluorenylidene)diphenol, a BPA substitute, which was recently used in BPA-free products, was also studied for its effect on cell viability and necrosis in a placental cell line. PAHs used in this experiment are as follows: 1. Anthracene (Sigma-Aldrich, St Louis, MO USA, #07, 671-100 mg) 2. Benzo[k]fluoranthene (Sigma-Aldrich, St Louis, MO USA, #03323-10 mg) 3. Benzo[a]pyrene (Sigma-Aldrich, St Louis, MO USA, #51968-50 mg) 4. 4,4-(9-Fluorenylidene)diphenol (BPA substitute) (Sigma-Aldrich, St Louis, MO USA, #39981-25 g) Cell culture and treatment The human placental HTR-8/SVneo cell line was provided by Queen’s University (Ontrio, Canada). HTR-8/SVneo cells were seeded in 100-mm culture plates at a density of 1 × 10 6 , followed by starvation for 24 h to increase the effectiveness of the substance to be treated. After the cells were grown up to 80% confluency in the plate containing RPMI 1640 medium without fetal bovine serum (Gibco, CAT NO. #21875034), the serum-free medium was removed, the cells were washed lightly with phosphate-buffered saline to remove all traces of the medium, and were then treated with trypsin/EDTA. After treatment with trypsin/EDTA for 1 min, the plate was tapped lightly on one side for the detachment of the cells from the bottom of the plate. After removing the cells, 10 mL of serum-free medium was evenly sprayed into the plate to remove all remaining cells from the bottom, and the solution containing the cells was collected, and transferred to a 15-mL Eppendorf tube. The solution containing the cells was centrifuged at 200 × g for 10 min (Centrifuge 5810r, Eppendorf). After centrifugation, we the supernatant was discarded and 4 mL of RPMI 1640 medium was added to the pellet. Then, 1 mL of cell suspension was transferred into a microtube, and the cells were detached once again, following which, 20 µL of the cell suspension was transferred into another microtube. After the sufficient mixing of the cell suspension, 20 µL of the cell suspension was injected into the narrow groove of the prepared hemocytometer using a pipette. We counted the cells in 4 squares of the hemocytometer mounted on a microscope at ×10 magnification. The diluted cells of 100 µL each were transferred in three compartments of 96-well microtiter plates in triplicate. Only the cell-free medium (100 µL) was separately added into the three compartments and was used as a blank. The seeded plates were incubated for 12 h at 37°C in 5% CO 2 . After confirming that the cells were attached to the plate using a microscope, the cells were treated with each PAH separately and cultured. Dose response Prior to the experiment, we attempted to determine the effective concentration of each PAH to use in time-response experiments with PAHs. The prepared cells were incubated for 24 h at predetermined concentrations (Table 1 ) [ 8 – 10 ]. After dissolving PAHs in dimethyl sulfoxide, they were diluted with RPMI 1640 medium at different concentrations. The cells were cultured with 0 µg/mL, 0.05 µg/mL, 0.5 µg/mL, 5 µg/mL, and 50 µg/mL of anthrane; 0 µg/mL, 0.3 µg/mL, 3 µg/mL, 30 µg/mL, and 300 µg/mL of benzo[k]fluoranthene; 0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL of benzo[a]pyrene; 0 µg/mL, 0.005 µg/mL, 0.05 µg/mL, 0.5 µg/mL, and 5 µg/mL of 4,4-(9-Fluorenylidene)diphenol. Cell growth and viability were examined by measuring absorbance using the XTT assay (XTT Cell Proliferation Assay Kit, American Type Culture Collection, Manassas, VA, USA) at 450–650 nm. The concentration of PAH showing a statistically significant change in optical density (OD) was compared with the respective control. Table 1 Absorbance of HTR8/SVneo according to the exposure dose of the endocrine disruptors Materials Doses (µg/mL) O.D. (Individuals) O.D. (Total, n = 4) Anthracene 0 1.502 1.534 1.572 1.510 1.530 ± 0.029 0.05 1.500 1.520 1.566 1.475 1.515 ± 0.036 0.5 1.469 1.501 1.508 1.400 1.470 ± 0.046 * 5 1.414 1.469 1.453 1.345 1.420 ± 0.051 ** 50 1.385 1.419 1.399 1.260 1.366 ± 0.067 ** Benzo[k]fluoranthene (B[k]F) 0 1.543 1.555 1.570 1.552 1.555 ± 0.010 0.3 1.569 1.574 1.564 1.560 1.567 ± 0.006 3 1.546 1.539 1.567 1.542 1.549 ± 0.012 30 1.540 1.524 1.549 1.539 1.538 ± 0.010 * 300 1.510 1.508 1.483 1.500 1.500 ± 0.011 ** Benzo[a]pyrene (B[a]P) 0 1.560 1.575 1.522 1.547 1.551 ± 0.021 0.1 1.505 1.592 1.500 1.541 1.535 ± 0.039 1 1.582 1.547 1.510 1.538 1.544 ± 0.027 10 1.481 1.502 1.494 1.504 1.495 ± 0.010 ** 100 1.205 1.464 1.470 1.429 1.392 ± 0.117 * Fluorene-9-bisphenol (BHPF) 0 1.541 1.509 1.512 1.572 1.534 ± 0.027 0.005 1.550 1.481 1.515 1.571 1.529 ± 0.037 0.05 1.521 1.479 1.473 1.599 1.518 ± 0.054 0.5 1.500 1.452 1.465 1.555 1.493 ± 0.043 5 1.401 1.429 1.391 1.462 1.421 ± 0.029 ** Total values are mean ± SEM. Significantly different from control; p < 0.05*, p < 0.005* Time response The most effective concentration confirmed for each PAH in the dose–response experiment was used to incubate the prepared cells for 24, 48, and 72 h (5% CO 2 , humidified atmosphere at 37°C). The OD value for each incubation time was determined using the XTT assay. XTT assay An XTT solution was prepared by dissolving 1 mL of activation reagent (sterile solution containing N-methyl dibenzopyrazine methyl sulfate) with 5 mL of XTT reagent at 37°C immediately before use. Then, 50 µL of the solution was added to each well, incubated at 37°C in a CO 2 incubator for 3 h, and the plate was slowly shaken manually until the solution turned orange. The absorbance of the wells containing cells and blank background control was measured at 450–500 nm using a microtiter plate reader. The absorbance of the cell-containing wells and control wells was also measured at 630–690 nm to assess non-specific readings. We determined the average value from the triplicate readings and subtracted the average value for the blank wells as well as the average value of the non-specific readings. When performing the XTT assay, the following parameters were used: Specific absorbance filter: 475 nm Non-Specific absorbance filter: 660 nm The specific absorbance of the sample was calculated using the following formula: Specific absorbance = A 475nm (Test) − A 475nm (Blank) − A 660nm (Test) Statistical analysis Each experimental group was conducted with the minimum number (n = 3) required for statistical analysis. The results were analyzed using t-tests; p < 0.05 was considered statistically significant. Results Dose response OD was measured at 450–650 nm. The absorbance before exposure to anthracene was 1.530 ± 0.029, but as the concentration increased, the OD value decreased. For anthracene at 0.5 µg/mL concentration, the OD was 1.470 ± 0.046, which was significantly lower than that before treatment (p < 0.05). When the cells were treated with 5 and 50 µg/mL anthracene, the OD values were also significantly lower than before the treatment (1.530 ± 0.029 vs. 1.420 ± 0.051, p < 0.005; 1.530 ± 0.029 vs. 1.366 ± 0.067, p < 0.005, respectively). In HTR-8/SVneo cells treated with increasing benzo[k]fluoranthene concentrations (3 µg/mL), the absorbance decreased with an increase in concentration. When the concentration reached 30 µg/mL, there was a significant difference as compared to the effect before treatment (1.555 ± 0.010 vs. 1.538 ± 0.010, p < 0.05, respectively). After treatment with 300 µg/mL benzo[k]fluoranthene, the absorbance was 1.500 ± 0.011, which was significantly lower than that of the control (p < 0.005). With benzo[a]pyrene concentration of 10 µg/mL and 100 µg/mL, the absorbance was significantly lower than that of the control (1.551 ± 0.021 vs. 1.495 ± 0.010, p < 0.005; 1.551 ± 0.021 vs. 1.392 ± 0.117, p < 0.05, respectively). Treatment with 4,4-(9-fluorenylidene)diphenol, which is a substitute for BPA, showed a significant difference in absorbance when treated with 5 µg/mL (1.534 ± 0.027 vs. 1.421 ± 0.029, p < 0.005) (Table 1 and Figure 1 ). Time response The change in absorbance over time was confirmed by treatment with the endocrine disruptor at the dose verified in the dose–response experiment (Table 2 and Figure 2 ). The initial absorbance obtained following the treatment of HTR-8/SVneo cells with anthracene (50 µg/mL) was 1.517 ± 0.011, which showed a tendency to decrease to 1.402 ± 0.068 (p < 0.05) at 24 h, 1.097 ± 0.132 (p < 0.05) at 48 h, and 0.568 ± 0.141 (p < 0.005) at 72 h. The absorbance following treatment with 300 µg/mL of benzo[k]fluoranthene was also significantly lower than the initial value at 24, 48, and 72 h (1.545 ± 0.025 vs. 1.513 ± 0.010, p < 0.05; 1.545 ± 0.025 vs. 1.495 ± 0.017, p < 0.05; 1.545 ± 0.025 vs. 1.483 ± 0.015, p < 0.005). The absorbance for the treatment with benzo[a]pyrene at a concentration of 100 µg/mL, as confirmed by the XTT assay, showed a significant difference after 24 h (1.553 ± 0.023 vs. 1.426 ± 0.056, p < 0.005; 1.553 ± 0.023 vs. 1.139 ± 0.072, p < 0.005; 1.553 ± 0.023 vs. 0.730 ± 0.020, p < 0.005). Similar results were also observed when the cells were treated with 5 µg/mL of 4,4-(9-fluorenylidene)diphenol. When compared with the initial OD value, the absorbance was statistically significantly lower than that of the control at 24 h, 48 h, and 72 h (1.539 ± 0.025 vs. 1.419 ± 0.022, p < 0.005; 1.539 ± 0.025 vs. 1.376 ± 0.039, p < 0.005; 1.539 ± 0.025 vs. 1.236 ± 0.089, p < 0.005). Table 2 Absorbance of HTR8/SVneo according to time course Materials Time course (h) OD (Individuals) OD (Total, n = 4) Anthracene (50 µg/mL) 0 1.502 1.511 1.532 1.506 1.517 ± 0.011 24 1.373 1.451 1.470 1.312 1.402 ± 0.068 * 48 1.042 1.285 1.115 0.947 1.097 ± 0.132 ** 72 0.495 0.702 0.388 0.685 0.568 ± 0.141 ** Benzo[k]fluoranthene (B[k]F) (300 µg/mL) 0 1.562 1.519 1.563 1.524 1.545 ± 0.025 24 1.522 1.500 1.521 1.509 1.513 ± 0.010 * 48 1.509 1.471 1.510 1.490 1.495 ± 0.017 * 72 1.478 1.469 1.507 1.477 1.483 ± 0.015 ** Benzo[a]pyrene (B[a]P) (100 µg/mL) 0 1.582 1.560 1.549 1.522 1.553 ± 0.023 24 1.450 1.441 1.475 1.338 1.426 ± 0.056 ** 48 1.115 1.230 1.046 1.165 1.139 ± 0.072 ** 72 0.701 0.726 0.749 0.745 0.730 ± 0.020 ** Fluorene-9-bisphenol (BHPF) (5 µg/mL) 0 1.502 1.550 1.565 1.540 1.539 ± 0.025 24 1.411 1.453 1.400 1.410 1.419 ± 0.022 ** 48 1.380 1.414 1.317 1.392 1.376 ± 0.039 ** 72 1.225 1.375 1.186 1.159 1.236 ± 0.089 ** All values are mean ± SEM. Significantly different from control; p < 0.05*, p < 0.005**. Discussion In this study, we evaluated changes in the placental cell line treated with PAHs to determine the effects of PAHs on cell viability and necrosis. We found that the absorbance measured using the XTT assay decreased with an increase in the concentrations of PAHs and time in HTR-8/SVneo cells treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4′-(9-fluorenylidene)diphenol. This result indicates a positive correlation between the above PAHs and cell necrosis according to the dose and time in the placental cell line. The placenta plays an important role in nutrient transport and fetal growth during pregnancy. The formation of a spiral artery from the placenta provides an uninterrupted supply of nutrient-rich maternal blood to the fetus by the end of the first trimester [ 11 ]. Human placental growth hormone, human chorionic gonadotropin, progesterone, estradiol, and placenta lactogen are secreted from the human placenta; all play a crucial role in pregnancy and fetal development [ 12 ]. Several studies have reported that exposure to PAHs in pregnant women may be associated with many changes in fetal and pregnancy outcomes, including pregnancy loss, changes in the onset of labor, such as preterm delivery, and abnormal fetal growth. In addition, it has been reported that the placenta may play a mediating role in these endocrine disruptors during pregnancy [ 13 – 17 ]. Our results showed that the placenta exposed to PAHs may be adversely affected, leading to complications during pregnancy. In particular, the loss of cell viability in the placenta is associated with premature labor. We provide experimental evidence to prove that PAHs induce preterm labor by confirming that PAHs evoke necrosis of placental cells. In a study investigating the correlation between the PAH concentration in the placenta and preterm labor in women exposed to high PAH exposure, it was found that the concentration and DNA products of PAHs affect preterm labor. They reported that the concentrations of benzo[a]pyrene, benzo[b]fluorene, and dibenzo[a,h]anthracene were higher in the placenta of pregnant women with preterm delivery than in those with full-term delivery [ 18 ]. PAHs are stabilizers and plasticizers found in many consumer products, including toys and hygiene products. They are also used in blood bags, medical devices, adhesives, solvents, and pesticides [ 19 , 20 ]. BPA and phthalate metabolites are detected in urinalysis in more than 90% of pregnant women in the United States because of their high PAH exposure [ 21 ]. However, the effects of endocrine disruptors on the placenta have not been studied despite pregnant women being exposed to endocrine disruptors almost daily. In addition, studies have focused on BPA. Exposure to PAHs during pregnancy is an essential issue in teratogenicity and preterm birth, and adverse perinatal outcome continues to increase. Our study is meaningful in that it provides the basis for research on the correlation between PAHs and preterm birth by providing evidence that PAH exposure causes placental cell necrosis. Furthermore, our study showed an important finding that BPA substitutes are also unsafe PAHs. Conclusions Our study showed that anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4′-(9-fluorenylidene)diphenol, a BPA substitute that has been insufficiently studied, affect cell proliferation and differentiation in a placental cell line. This means that these PAHs cause cell necrosis in the placenta, leading to adverse perinatal outcomes, such as preterm birth. These results can be used as basic data for future research related to preterm birth and PAHs. Abbreviations PAH, Polycyclic aromatic hydrocarbons BPA, bisphenol A OD, optical density Declarations Ethics approval Not applicable Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Funding The authors wish to acknowledge the financial support of the Catholic Medical Center Research Foundation in the program year of 2018. Authors’ contributions YSJ and HSK are the first authors participating in the study design and contributed equally to this work. AYK performed the experiments and analyzed the data. HGJ participated in the experiment. WJK participated in data interpretation. SKC is the corresponding author and participated in the design of the study, interpretation of the data, and drafting of the manuscript. All authors have read and approved the final manuscript. Acknowledgments Not applicable. References Sanderson JT. 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Int J Hyg Environ Health. 2007;210:623–34. Cite Share Download PDF Status: Published Journal Publication published 08 Mar, 2022 Read the published version in Reproductive Biology and Endocrinology → Version 1 posted Reviewer # 2 agreed at journal 19 Nov, 2021 Reviews received at journal 06 Nov, 2021 Reviewer # 1 agreed at journal 05 Nov, 2021 Reviewers invited by journal 03 Nov, 2021 Editor assigned by journal 03 Nov, 2021 Editor invited by journal 02 Nov, 2021 Submission checks completed at journal 30 Oct, 2021 First submitted to journal 26 Oct, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1019582","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":60236826,"identity":"49f6b0c3-f68c-4870-ad70-354a39d8c5ca","order_by":0,"name":"Yun Sung Jo","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yun","middleName":"Sung","lastName":"Jo","suffix":""},{"id":60236827,"identity":"2424f98a-a075-4b71-9494-cc3315a98c70","order_by":1,"name":"Hyun Sun Ko","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Sun","lastName":"Ko","suffix":""},{"id":60236828,"identity":"f5a61634-1be2-4f0f-b986-aa8392980688","order_by":2,"name":"Ah Young Kim","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ah","middleName":"Young","lastName":"Kim","suffix":""},{"id":60236829,"identity":"4e2aecdb-7f7c-4b5c-beaf-2c78ec873f48","order_by":3,"name":"Ha Gyeong Jo","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ha","middleName":"Gyeong","lastName":"Jo","suffix":""},{"id":60236830,"identity":"40ec693b-6276-4b2b-ae70-b6998461eddc","order_by":4,"name":"Woo Jung Kim","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Woo","middleName":"Jung","lastName":"Kim","suffix":""},{"id":60236831,"identity":"e4bd7e86-2ed6-4464-865a-ce507a0fbc7d","order_by":5,"name":"Sae Kyung Choi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACCRBxwAZI8DAwPDAAizEToyWNgYENqCWBBC2HoVoYiNDCL91+TfLHmfPy/PN7Dz5IKLBj4G8/wGxcgUeL5JwzZdI8N24bzjjGl2yQYJDMIHEmgTnxDB4tBjdy0qQZPtxOYDjGYyaRYHCAgeEGA/PBBgJaJH98OJcgD9MiT1hL+jEJnhsHEgxgWgyAWhLxaQH6hdma50yy4cZjOcYgv/AYnklsNsSnBRhiD2/+OGYnL3f4jOGDD3/s5OSOHz4siU8LMNJNJFC4DAyM+DUwMLA//kBAxSgYBaNgFIx0AAAodE18cSJrEgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-6264-4256","institution":"The Catholic University of Korea","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Sae","middleName":"Kyung","lastName":"Choi","suffix":""}],"badges":[],"createdAt":"2021-10-26 09:47:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1019582/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1019582/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12958-022-00920-7","type":"published","date":"2022-03-08T09:53:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":15098277,"identity":"6abdd272-534c-40a5-9260-172d3967b799","added_by":"auto","created_at":"2021-11-01 16:00:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40675,"visible":true,"origin":"","legend":"Dose response measured by the XTT assay (OD at 450–650 nm). HTR-8/SVneo cells were treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4-(9-fluorenylidene)diphenol for the cell viability analysis using XTT assay. The absorbance prior to anthracene exposure was 1.530 ± 0.029, which decreased with an increase in anthracene concentration. In HTR-8/SVneo cells treated with increasing concentrations of benzo[k]fluoranthene, the absorbance decreased with an increase in the concentration. Treatment with 300 µg/mL benzo[k]fluoranthene resulted in an OD of 1.500 ± 0.011, which was lower than that of the control. Following treatment with benzo[a]pyrene at concentrations of 10 µg/mL and 100 µg/mL, the absorbance was lower than that of the control. Treatment with 4,4-(9-fluorenylidene)diphenol, which is a substitute for BPA, showed a decrease in absorbance at a concentration of 5 µg/mL .","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1019582/v1/75a3e546eedfdec5f8e93bc0.png"},{"id":15098276,"identity":"21d948a1-6672-4b25-921f-d59c29eea76a","added_by":"auto","created_at":"2021-11-01 16:00:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13757,"visible":true,"origin":"","legend":"Time course in the XTT assay (OD at 450–650 nm). HTR-8/SVneo cells were treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4-(9-fluorenylidene)diphenol at 24, 48, and 72 h for the time course analysis using XTT assay. Treatment of the cells with anthracene (50 µg/mL) showed an initial absorbance of 1.517 ± 0.011, which showed a decreasing tendency with increasing time, decreasing to 1.402 ± 0.068 at 24 h, 1.097 ± 0.132 at 48 h, and 0.568 ± 0.141 at 72 h. The OD values obtained following 300 µg/mL benzo[k]fluoranthene treatment for 24, 48, and 72 h were also lower than the initial value. The absorbance following treatment with benzo [a]pyrene at a concentration of 100 μg/mL showed a decrease after 24 h. Similar results were observed when the cells were treated with 5 µg/mL of 4,4-(9-fluorenylidene)diphenol. Compared with the initial OD value, the absorbance was lower than that of the control at 24 h, 48 h, and 72 h.","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1019582/v1/975b30c9dd03650df4eaaebd.png"},{"id":18971602,"identity":"c47f2744-2eb4-4d79-ba4c-a0ba19777231","added_by":"auto","created_at":"2022-03-08 09:53:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":476789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1019582/v1/d4544948-09f2-48b0-833b-4b780a835181.pdf"}],"financialInterests":"","formattedTitle":"Effects of polycyclic aromatic hydrocarbons on the proliferation and differentiation of placental cells","fulltext":[{"header":"Background","content":"\u003cp\u003eEndocrine disruptors are chemicals that can disrupt the endocrine system in both humans and animals. Female reproductive ability is an important process regulated by hormones and is susceptible to exposure to endocrine-disrupting substances. Endocrine disruptors have a benzene ring structure similar to that of steroid hormones among several female hormones. Therefore, they can easily affect reproductive hormones with a benzene ring structure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Female reproductive disorders caused by endocrine disruptors can lead to inadequate hormone production, infertility, anovulation, premature ovarian failure, and menstrual disorders. Various endocrine disruptors have been reported to disrupt the endocrine system in reproductive-aged women and to cause congenital disabilities [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePolycyclic aromatic hydrocarbons (PAHs), which are endocrine disruptors, are common environmental pollutants released by incomplete combustion processes. PAHs emitted into our living environment are of interest because they have harmful side effects to humans, such as teratogenicity, mutagenicity, and carcinogenicity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. PAHs enter the body primarily through inhalation and ingestion. In pregnant women, PAHs that enter the body induce side effects on the mother, which are also transmitted through the placenta to the fetus, causing severe fetal developmental problems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The growth and function of the placenta are regulated and coordinated to perform the placental role efficiently during pregnancy. If PAHs are transmitted from the mother to the fetus through the placenta, they negatively affect fetal growth. In addition, exposure to harmful substances such as PAHs during pregnancy can lead to disability even after the fetus grows into an adult [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA recent study analyzed the correlation between preterm labor and the concentration of PAHs in the placenta of women living in areas with high PAH exposure. This study showed that high PAH concentration, DNA products exposed to PAHs, and regulation of endocrine metabolic pathways affect preterm labor in pregnant women living near areas contaminated with environmental pollutants.\u003c/p\u003e \u003cp\u003eGiven the results of these studies, exposure to PAHs during pregnancy is a critical issue in terms of teratogenicity, fetal growth, and preterm labor. However, there are various PAH types, and the studies so far have mainly focused on bisphenol A (BPA) and some phthalates. Therefore, we experimented on the PAHs that have not been studied for their effects on pregnancy. We examined cell viability and necrosis in a placental cell line treated with the selected PAHs. This study indicates the serious effects of PAHs that negatively affect pregnancy but were previously unknown. Moreover, the study is a reference for the identification of harmful PAHs during pregnancy prognosis in women who are more susceptible to PAH exposure.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eFour PAHs, which have not been explored for their effects on pregnancy, were selected for experimentation on cell viability and necrosis in a placental cell line treated with the selected PAHs. In addition, 4,4-9(fluorenylidene)diphenol, a BPA substitute, which was recently used in BPA-free products, was also studied for its effect on cell viability and necrosis in a placental cell line.\u003c/p\u003e \u003cp\u003ePAHs used in this experiment are as follows:\u003c/p\u003e\u003cp\u003e1. Anthracene (Sigma-Aldrich, St Louis, MO USA, #07, 671-100 mg)\u003c/p\u003e\n\u003cp\u003e2. Benzo[k]fluoranthene (Sigma-Aldrich, St Louis, MO USA, #03323-10 mg)\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e3. Benzo[a]pyrene (Sigma-Aldrich, St Louis, MO USA, #51968-50 mg)\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e4. 4,4-(9-Fluorenylidene)diphenol (BPA substitute) (Sigma-Aldrich, St Louis, MO USA, #39981-25 g)\u003c/p\u003e\n\u003c/span\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eThe human placental HTR-8/SVneo cell line was provided by Queen\u0026rsquo;s University (Ontrio, Canada). HTR-8/SVneo cells were seeded in 100-mm culture plates at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e, followed by starvation for 24 h to increase the effectiveness of the substance to be treated. After the cells were grown up to 80% confluency in the plate containing RPMI 1640 medium without fetal bovine serum (Gibco, CAT NO. #21875034), the serum-free medium was removed, the cells were washed lightly with phosphate-buffered saline to remove all traces of the medium, and were then treated with trypsin/EDTA. After treatment with trypsin/EDTA for 1 min, the plate was tapped lightly on one side for the detachment of the cells from the bottom of the plate. After removing the cells, 10 mL of serum-free medium was evenly sprayed into the plate to remove all remaining cells from the bottom, and the solution containing the cells was collected, and transferred to a 15-mL Eppendorf tube. The solution containing the cells was centrifuged at 200 \u0026times; g for 10 min (Centrifuge 5810r, Eppendorf). After centrifugation, we the supernatant was discarded and 4 mL of RPMI 1640 medium was added to the pellet. Then, 1 mL of cell suspension was transferred into a microtube, and the cells were detached once again, following which, 20 \u0026micro;L of the cell suspension was transferred into another microtube. After the sufficient mixing of the cell suspension, 20 \u0026micro;L of the cell suspension was injected into the narrow groove of the prepared hemocytometer using a pipette. We counted the cells in 4 squares of the hemocytometer mounted on a microscope at \u0026times;10 magnification. The diluted cells of 100 \u0026micro;L each were transferred in three compartments of 96-well microtiter plates in triplicate. Only the cell-free medium (100 \u0026micro;L) was separately added into the three compartments and was used as a blank. The seeded plates were incubated for 12 h at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. After confirming that the cells were attached to the plate using a microscope, the cells were treated with each PAH separately and cultured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDose response\u003c/h2\u003e \u003cp\u003ePrior to the experiment, we attempted to determine the effective concentration of each PAH to use in time-response experiments with PAHs. The prepared cells were incubated for 24 h at predetermined concentrations (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. After dissolving PAHs in dimethyl sulfoxide, they were diluted with RPMI 1640 medium at different concentrations. The cells were cultured with 0 \u0026micro;g/mL, 0.05 \u0026micro;g/mL, 0.5 \u0026micro;g/mL, 5 \u0026micro;g/mL, and 50 \u0026micro;g/mL of anthrane; 0 \u0026micro;g/mL, 0.3 \u0026micro;g/mL, 3 \u0026micro;g/mL, 30 \u0026micro;g/mL, and 300 \u0026micro;g/mL of benzo[k]fluoranthene; 0 \u0026micro;g/mL, 0.1 \u0026micro;g/mL, 1 \u0026micro;g/mL, 10 \u0026micro;g/mL, and 100 \u0026micro;g/mL of benzo[a]pyrene; 0 \u0026micro;g/mL, 0.005 \u0026micro;g/mL, 0.05 \u0026micro;g/mL, 0.5 \u0026micro;g/mL, and 5 \u0026micro;g/mL of 4,4-(9-Fluorenylidene)diphenol. Cell growth and viability were examined by measuring absorbance using the XTT assay (XTT Cell Proliferation Assay Kit, American Type Culture Collection, Manassas, VA, USA) at 450\u0026ndash;650 nm. The concentration of PAH showing a statistically significant change in optical density (OD) was compared with the respective control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAbsorbance of HTR8/SVneo according to the exposure dose of the endocrine disruptors\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDoses (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eO.D. (Individuals)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eO.D. (Total, n = 4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eAnthracene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.530 \u0026plusmn; 0.029\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.566\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.515 \u0026plusmn; 0.036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.508\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.470 \u0026plusmn; 0.046\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.345\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.420 \u0026plusmn; 0.051\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.385\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.366 \u0026plusmn; 0.067\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eBenzo[k]fluoranthene (B[k]F)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.555 \u0026plusmn; 0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.564\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.567 \u0026plusmn; 0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.567\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.549 \u0026plusmn; 0.012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.538 \u0026plusmn; 0.010\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.508\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.500 \u0026plusmn; 0.011\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eBenzo[a]pyrene (B[a]P)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.551 \u0026plusmn; 0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.535 \u0026plusmn; 0.039\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.544 \u0026plusmn; 0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.494\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.495 \u0026plusmn; 0.010\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.464\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.392 \u0026plusmn; 0.117\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eFluorene-9-bisphenol (BHPF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.541\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.534 \u0026plusmn; 0.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.481\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.515\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.529 \u0026plusmn; 0.037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.518 \u0026plusmn; 0.054\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.465\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.493 \u0026plusmn; 0.043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.391\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.421 \u0026plusmn; 0.029\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eTotal values are mean \u0026plusmn; SEM.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eSignificantly different from control; p \u0026lt; 0.05*, p \u0026lt; 0.005*\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTime response\u003c/h2\u003e \u003cp\u003eThe most effective concentration confirmed for each PAH in the dose\u0026ndash;response experiment was used to incubate the prepared cells for 24, 48, and 72 h (5% CO\u003csub\u003e2\u003c/sub\u003e, humidified atmosphere at 37\u0026deg;C). The OD value for each incubation time was determined using the XTT assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eXTT assay\u003c/h2\u003e \u003cp\u003eAn XTT solution was prepared by dissolving 1 mL of activation reagent (sterile solution containing N-methyl dibenzopyrazine methyl sulfate) with 5 mL of XTT reagent at 37\u0026deg;C immediately before use. Then, 50 \u0026micro;L of the solution was added to each well, incubated at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator for 3 h, and the plate was slowly shaken manually until the solution turned orange. The absorbance of the wells containing cells and blank background control was measured at 450\u0026ndash;500 nm using a microtiter plate reader. The absorbance of the cell-containing wells and control wells was also measured at 630\u0026ndash;690 nm to assess non-specific readings. We determined the average value from the triplicate readings and subtracted the average value for the blank wells as well as the average value of the non-specific readings. When performing the XTT assay, the following parameters were used:\u003c/p\u003e \u003cp\u003eSpecific absorbance filter: 475 nm\u003c/p\u003e \u003cp\u003eNon-Specific absorbance filter: 660 nm\u003c/p\u003e \u003cp\u003eThe specific absorbance of the sample was calculated using the following formula:\u003c/p\u003e \u003cp\u003eSpecific absorbance = A\u003csub\u003e475nm\u003c/sub\u003e (Test) \u0026minus; A\u003csub\u003e475nm\u003c/sub\u003e (Blank) \u0026minus; A\u003csub\u003e660nm\u003c/sub\u003e (Test)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEach experimental group was conducted with the minimum number (n = 3) required for statistical analysis. The results were analyzed using t-tests; p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDose response\u003c/h2\u003e \u003cp\u003eOD was measured at 450\u0026ndash;650 nm. The absorbance before exposure to anthracene was 1.530 \u0026plusmn; 0.029, but as the concentration increased, the OD value decreased. For anthracene at 0.5 \u0026micro;g/mL concentration, the OD was 1.470 \u0026plusmn; 0.046, which was significantly lower than that before treatment (p \u0026lt; 0.05). When the cells were treated with 5 and 50 \u0026micro;g/mL anthracene, the OD values were also significantly lower than before the treatment (1.530 \u0026plusmn; 0.029 vs. 1.420 \u0026plusmn; 0.051, p \u0026lt; 0.005; 1.530 \u0026plusmn; 0.029 vs. 1.366 \u0026plusmn; 0.067, p \u0026lt; 0.005, respectively).\u003c/p\u003e \u003cp\u003eIn HTR-8/SVneo cells treated with increasing benzo[k]fluoranthene concentrations (3 \u0026micro;g/mL), the absorbance decreased with an increase in concentration. When the concentration reached 30 \u0026micro;g/mL, there was a significant difference as compared to the effect before treatment (1.555 \u0026plusmn; 0.010 vs. 1.538 \u0026plusmn; 0.010, p \u0026lt; 0.05, respectively). After treatment with 300 \u0026micro;g/mL benzo[k]fluoranthene, the absorbance was 1.500 \u0026plusmn; 0.011, which was significantly lower than that of the control (p \u0026lt; 0.005).\u003c/p\u003e \u003cp\u003eWith benzo[a]pyrene concentration of 10 \u0026micro;g/mL and 100 \u0026micro;g/mL, the absorbance was significantly lower than that of the control (1.551 \u0026plusmn; 0.021 vs. 1.495 \u0026plusmn; 0.010, p \u0026lt; 0.005; 1.551 \u0026plusmn; 0.021 vs. 1.392 \u0026plusmn; 0.117, p \u0026lt; 0.05, respectively). Treatment with 4,4-(9-fluorenylidene)diphenol, which is a substitute for BPA, showed a significant difference in absorbance when treated with 5 \u0026micro;g/mL (1.534 \u0026plusmn; 0.027 vs. 1.421 \u0026plusmn; 0.029, p \u0026lt; 0.005) (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTime response\u003c/h2\u003e \u003cp\u003eThe change in absorbance over time was confirmed by treatment with the endocrine disruptor at the dose verified in the dose\u0026ndash;response experiment (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The initial absorbance obtained following the treatment of HTR-8/SVneo cells with anthracene (50 \u0026micro;g/mL) was 1.517 \u0026plusmn; 0.011, which showed a tendency to decrease to 1.402 \u0026plusmn; 0.068 (p \u0026lt; 0.05) at 24 h, 1.097 \u0026plusmn; 0.132 (p \u0026lt; 0.05) at 48 h, and 0.568 \u0026plusmn; 0.141 (p \u0026lt; 0.005) at 72 h. The absorbance following treatment with 300 \u0026micro;g/mL of benzo[k]fluoranthene was also significantly lower than the initial value at 24, 48, and 72 h (1.545 \u0026plusmn; 0.025 vs. 1.513 \u0026plusmn; 0.010, p \u0026lt; 0.05; 1.545 \u0026plusmn; 0.025 vs. 1.495 \u0026plusmn; 0.017, p \u0026lt; 0.05; 1.545 \u0026plusmn; 0.025 vs. 1.483 \u0026plusmn; 0.015, p \u0026lt; 0.005). The absorbance for the treatment with benzo[a]pyrene at a concentration of 100 \u0026micro;g/mL, as confirmed by the XTT assay, showed a significant difference after 24 h (1.553 \u0026plusmn; 0.023 vs. 1.426 \u0026plusmn; 0.056, p \u0026lt; 0.005; 1.553 \u0026plusmn; 0.023 vs. 1.139 \u0026plusmn; 0.072, p \u0026lt; 0.005; 1.553 \u0026plusmn; 0.023 vs. 0.730 \u0026plusmn; 0.020, p \u0026lt; 0.005). Similar results were also observed when the cells were treated with 5 \u0026micro;g/mL of 4,4-(9-fluorenylidene)diphenol. When compared with the initial OD value, the absorbance was statistically significantly lower than that of the control at 24 h, 48 h, and 72 h (1.539 \u0026plusmn; 0.025 vs. 1.419 \u0026plusmn; 0.022, p \u0026lt; 0.005; 1.539 \u0026plusmn; 0.025 vs. 1.376 \u0026plusmn; 0.039, p \u0026lt; 0.005; 1.539 \u0026plusmn; 0.025 vs. 1.236 \u0026plusmn; 0.089, p \u0026lt; 0.005).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAbsorbance of HTR8/SVneo according to time course\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTime course (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003eOD (Individuals)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eOD (Total, n = 4)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAnthracene\u003c/p\u003e \u003cp\u003e(50 \u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.517 \u0026plusmn; 0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.373\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.402 \u0026plusmn; 0.068\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.285\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.097 \u0026plusmn; 0.132\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.495\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.388\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.568 \u0026plusmn; 0.141\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eBenzo[k]fluoranthene (B[k]F)\u003c/p\u003e \u003cp\u003e(300 \u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.545 \u0026plusmn; 0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.521\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.513 \u0026plusmn; 0.010\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.471\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.495 \u0026plusmn; 0.017\u003csup\u003e\u003cb\u003e*\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.478\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.477\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.483 \u0026plusmn; 0.015\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eBenzo[a]pyrene (B[a]P)\u003c/p\u003e \u003cp\u003e(100 \u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.582\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.549\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.522\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.553 \u0026plusmn; 0.023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.338\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.426 \u0026plusmn; 0.056\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.139 \u0026plusmn; 0.072\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.726\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.749\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.745\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.730 \u0026plusmn; 0.020\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eFluorene-9-bisphenol (BHPF)\u003c/p\u003e \u003cp\u003e(5 \u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.539 \u0026plusmn; 0.025\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.419 \u0026plusmn; 0.022\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.392\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.376 \u0026plusmn; 0.039\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e1.236 \u0026plusmn; 0.089\u003csup\u003e\u003cb\u003e**\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAll values are mean \u0026plusmn; SEM. Significantly different from control; p \u0026lt; 0.05*, p \u0026lt; 0.005**.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated changes in the placental cell line treated with PAHs to determine the effects of PAHs on cell viability and necrosis. We found that the absorbance measured using the XTT assay decreased with an increase in the concentrations of PAHs and time in HTR-8/SVneo cells treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4\u0026prime;-(9-fluorenylidene)diphenol. This result indicates a positive correlation between the above PAHs and cell necrosis according to the dose and time in the placental cell line.\u003c/p\u003e \u003cp\u003eThe placenta plays an important role in nutrient transport and fetal growth during pregnancy. The formation of a spiral artery from the placenta provides an uninterrupted supply of nutrient-rich maternal blood to the fetus by the end of the first trimester [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Human placental growth hormone, human chorionic gonadotropin, progesterone, estradiol, and placenta lactogen are secreted from the human placenta; all play a crucial role in pregnancy and fetal development [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Several studies have reported that exposure to PAHs in pregnant women may be associated with many changes in fetal and pregnancy outcomes, including pregnancy loss, changes in the onset of labor, such as preterm delivery, and abnormal fetal growth. In addition, it has been reported that the placenta may play a mediating role in these endocrine disruptors during pregnancy [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Our results showed that the placenta exposed to PAHs may be adversely affected, leading to complications during pregnancy.\u003c/p\u003e \u003cp\u003eIn particular, the loss of cell viability in the placenta is associated with premature labor. We provide experimental evidence to prove that PAHs induce preterm labor by confirming that PAHs evoke necrosis of placental cells. In a study investigating the correlation between the PAH concentration in the placenta and preterm labor in women exposed to high PAH exposure, it was found that the concentration and DNA products of PAHs affect preterm labor. They reported that the concentrations of benzo[a]pyrene, benzo[b]fluorene, and dibenzo[a,h]anthracene were higher in the placenta of pregnant women with preterm delivery than in those with full-term delivery [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePAHs are stabilizers and plasticizers found in many consumer products, including toys and hygiene products. They are also used in blood bags, medical devices, adhesives, solvents, and pesticides [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. BPA and phthalate metabolites are detected in urinalysis in more than 90% of pregnant women in the United States because of their high PAH exposure [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the effects of endocrine disruptors on the placenta have not been studied despite pregnant women being exposed to endocrine disruptors almost daily. In addition, studies have focused on BPA.\u003c/p\u003e \u003cp\u003eExposure to PAHs during pregnancy is an essential issue in teratogenicity and preterm birth, and adverse perinatal outcome continues to increase. Our study is meaningful in that it provides the basis for research on the correlation between PAHs and preterm birth by providing evidence that PAH exposure causes placental cell necrosis. Furthermore, our study showed an important finding that BPA substitutes are also unsafe PAHs.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study showed that anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4\u0026prime;-(9-fluorenylidene)diphenol, a BPA substitute that has been insufficiently studied, affect cell proliferation and differentiation in a placental cell line. This means that these PAHs cause cell necrosis in the placenta, leading to adverse perinatal outcomes, such as preterm birth. These results can be used as basic data for future research related to preterm birth and PAHs.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003ePAH, Polycyclic aromatic hydrocarbons\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBPA, bisphenol A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOD, optical density\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics approval\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to acknowledge the financial support of the Catholic Medical Center Research Foundation in the program year of 2018.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYSJ and HSK are the first authors participating in the study design and contributed equally to this work. AYK performed the experiments and analyzed the data. HGJ participated in the experiment. WJK participated in data interpretation. SKC is the corresponding author and participated in the design of the study, interpretation of the data, and drafting of the manuscript. All authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSanderson JT. The steroid hormone biosynthesis pathway as a target for endocrine-disrupting chemicals. Toxicol Sci. 2006;94:3\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSweeney T. Is exposure to endocrine disrupting compounds during fetal/post-natal development affecting the reproductive potential of farm animals? Domest Anim Endocrinol. 2002;23:203\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel S, Zhou C, Rattan S, Flaws JA. Effects of Endocrine-Disrupting Chemicals on the Ovary. Biol Reprod. 2015;93:20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodruff TK, Walker CL. Fetal and early postnatal environmental exposures and reproductive health effects in the female. Fertil Steril. 2008;89:e47-51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBostr\u0026ouml;m CE, Gerde P, Hanberg A, Jernstr\u0026ouml;m B, Johansson C, Kyrklund T, et al. Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. Environ Health Perspect. 2002;110 Suppl 3:451\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller KP, Borgeest C, Greenfeld C, Tomic D, Flaws JA. In utero effects of chemicals on reproductive tissues in females. Toxicol Appl Pharmacol. 2004;198:111\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerera FP, Rauh V, Tsai WY, Kinney P, Camann D, Barr D, et al. Effects of transplacental exposure to environmental pollutants on birth outcomes in a multiethnic population. Environ Health Perspect. 2003;111:201\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim A, Park M, Yoon TK, Lee WS, Ko JJ, Lee K, et al. Maternal exposure to benzo[b]fluoranthene disturbs reproductive performance in male offspring mice. Toxicol Lett. 2011;203:54\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Vee M, Kolasa E, Jouan E, Collet N, Fardel O. Differentiation of human placental BeWo cells by the environmental contaminant benzo(a)pyrene. Chem Biol Interact. 2014;210:1\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrwal E, Rak A, Grochowalski A, Milewicz T, Gregoraszczuk EL. Cell-specific and dose-dependent effects of PAHs on proliferation, cell cycle, and apoptosis protein expression and hormone secretion by placental cell lines. Toxicol Lett. 2017;280:10\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRattan S, Zhou C, Chiang C, Mahalingam S, Brehm E, Flaws JA. Exposure to endocrine disruptors during adulthood: consequences for female fertility. J Endocrinol. 2017;233:R109-r29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod. 2003;69:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodruff TJ, Zota AR, Schwartz JM. Environmental chemicals in pregnant women in the United States: NHANES 2003-2004. Environ Health Perspect. 2011;119:878\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeiga-Lopez A, Kannan K, Liao C, Ye W, Domino SE, Padmanabhan V. Gender-Specific Effects on Gestational Length and Birth Weight by Early Pregnancy BPA Exposure. J Clin Endocrinol Metab. 2015;100:E1394-403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdibi JJ, Hauser R, Williams PL, Whyatt RM, Calafat AM, Nelson H, et al. Maternal urinary metabolites of Di-(2-Ethylhexyl) phthalate in relation to the timing of labor in a US multicenter pregnancy cohort study. Am J Epidemiol. 2009;169:1015\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerguson KK, McElrath TF, Ko YA, Mukherjee B, Meeker JD. Variability in urinary phthalate metabolite levels across pregnancy and sensitive windows of exposure for the risk of preterm birth. Environ Int. 2014;70:118\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelsenfeld G. A brief history of epigenetics. Cold Spring Harb Perspect Biol. 2014;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuter MA, Aagaard KM, Coarfa C, Robertson M, Zhou G, Jackson BP, et al. Association between elevated placental polycyclic aromatic hydrocarbons (PAHs) and PAH-DNA adducts from Superfund sites in Harris County, and increased risk of preterm birth (PTB). Biochem Biophys Res Commun. 2019;516:344\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMervish N, McGovern KJ, Teitelbaum SL, Pinney SM, Windham GC, Biro FM, et al. Dietary predictors of urinary environmental biomarkers in young girls, BCERP, 2004-7. Environ Res. 2014;133:12\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNewbern D, Freemark M. Placental hormones and the control of maternal metabolism and fetal growth. Curr Opin Endocrinol Diabetes Obes. 2011;18:409\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeudorf U, Mersch-Sundermann V, Angerer J. Phthalates: toxicology and exposure. Int J Hyg Environ Health. 2007;210:623\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pregnancy, Placenta, Endocrine disruptors, Anthracene, Female reproduction, Polycyclic aromatic hydrocarbons","lastPublishedDoi":"10.21203/rs.3.rs-1019582/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1019582/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons (PAHs) other than bisphenol A (BPA) and BPA substitutes on placental cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHTR-8/SVneo cells were treated with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, which is used as a substitute for BPA-free products. After confirming the dose response for each reagent using the prepared cells, the cells were incubated for 24, 48, and 72 h. Cell viability was confirmed using the XTT assay. Each experiment was performed with the minimum number of samples (n = 3) required for statistical analysis. The results were analyzed using t-tests; p \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAfter treatment with anthracene, benzo[k]fluoranthene, benzo[a]pyrene, endocrine disruptors, and 4,4-(9-fluorenylidene)diphenol, the absorbance measured using the XTT assay decreased significantly with increasing concentration. The absorbance decreased significantly over time following treatment with each endocrine disruptor at the concentration confirmed by the dose\u0026ndash;response analysis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study showed that anthracene, benzo[k]fluoranthene, benzo[a]pyrene, and 4,4-(9-fluorenylidene)diphenol\u0026mdash;a BPA substitute\u0026mdash;affect cell viability and necrosis in the placental cell line. The study indicates the serious effects of PAHs that negatively affect pregnancy but were previously unknown. Further, this study would serve as a reference for the identification of harmful PAHs during pregnancy prognosis in women who are more susceptible to PAH exposure.\u003c/p\u003e","manuscriptTitle":"Effects of polycyclic aromatic hydrocarbons on the proliferation and differentiation of placental cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-11-01 16:00:30","doi":"10.21203/rs.3.rs-1019582/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2021-11-20T00:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-11-06T20:07:41+00:00","index":0,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-11-06T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-11-03T12:32:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-11-03T04:46:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-11-02T23:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-10-30T21:19:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Reproductive Biology and Endocrinology","date":"2021-10-26T05:45:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"reproductive-biology-and-endocrinology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbej","sideBox":"Learn more about [Reproductive Biology and Endocrinology](http://rbej.biomedcentral.com)","snPcode":"12958","submissionUrl":"https://submission.nature.com/new-submission/12958/3","title":"Reproductive Biology and Endocrinology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b44a419e-d521-424f-bd55-072447eb99b2","owner":[],"postedDate":"November 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":8230224,"name":"Endocrinology \u0026 Metabolism"}],"tags":[],"updatedAt":"2022-03-08T09:53:10+00:00","versionOfRecord":{"articleIdentity":"rs-1019582","link":"https://doi.org/10.1186/s12958-022-00920-7","journal":{"identity":"reproductive-biology-and-endocrinology","isVorOnly":false,"title":"Reproductive Biology and Endocrinology"},"publishedOn":"2022-03-08 09:53:10","publishedOnDateReadable":"March 8th, 2022"},"versionCreatedAt":"2021-11-01 16:00:30","video":"","vorDoi":"10.1186/s12958-022-00920-7","vorDoiUrl":"https://doi.org/10.1186/s12958-022-00920-7","workflowStages":[]},"version":"v1","identity":"rs-1019582","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1019582","identity":"rs-1019582","version":["v1"]},"buildId":"iFTdqyg4nuje_uAy1AHro","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}